Real-time monitoring of angiotensin II-induced contractile response and cytoskeleton remodeling in individual cells by atomic force microscopy.
Physiological processes, occurring as a result of specific receptor stimulation, are generally assessed via molecular biology techniques and microscopic approaches with the involvement of specific molecular markers. The recent progress in experimental approaches, allowing the mechanical characterization of individual biological entities, now makes it possible to address cellular processes occurring in individual cells as a result of their stimulation by hormones. Here, we demonstrate that the atomic force microscope (AFM) can be used to mechanically probe individual cells following the activation of the angiotensin-1 receptor, a receptor well known for its role in cell homeostasis regulation. Our goal is to demonstrate that the measurement of cantilever deflection can be used to quantify in real time the mechanical and morphological cell activity associated with the activation of the receptor. By combining the AFM with time-lapse sequences of phase-contrast and confocal micrographs, we show that the angiotensin-1 receptor stimulation with 100 nM angiotensin II produces an actin-dependent contractile response with an amplitude of 262 +/- 52 nm. We validated the mechanical origin of the responses by measuring the elastic modulus of the cell from indentation experiments performed at 30-s intervals. Additionally, nanoscaled height fluctuations of the cell membrane occurring after the initial contraction response could be attributed to an increased actin cytoskeleton activity and remodeling detected by confocal microscopy. Finally, by using inhibitors for specific elements of the angiotensin-1 receptor signaling pathways, we demonstrate that AFM real-time height monitoring allows a read out of the molecular processes responsible for the cell mechanical response.
Cuerrier
Charles M
CM
Département de Pharmacologie, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada.
Benoit
Martin
M
Guillemette
Gaétan
G
Gobeil
Fernand
F
Grandbois
Michel
M
D000199:Actins / Q000502:physiology*; D000577:Amides / Q000494:pharmacology; D000804:Angiotensin II / Q000502:physiology*; D001381:Azepines / Q000494:pharmacology; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D003584:Cytological Techniques; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D055119:Elastic Modulus; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000196:embryology; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D044140:Receptor, Angiotensin, Type 1 / Q000187:drug effects / Q000502:physiology*
D000199:Actins; D000577:Amides; D001381:Azepines; D006576:Heterocyclic Compounds, 4 or More Rings; D011725:Pyridines; D044140:Receptor, Angiotensin, Type 1; C056218:ML 9; D000804:Angiotensin II; C108830:Y 27632; C472645:blebbistatin